WO2017057450A1 - Peptide that specifically accumulates in biliary tract cancer, and use thereof - Google Patents

Abstract

The present invention provides a novel peptide that specifically accumulates in biliary tract cancer. The peptide according to the present invention is (a) a peptide having an amino acid sequence containing a sequence represented by SEQ ID NO. 1, or (b) a peptide having an amino acid sequence containing a sequence having an identity of 60% or more with respect to the sequence represented by SEQ ID NO. 1, and specifically accumulates in biliary tract cancer.

Description

Peptide having specific accumulation property for biliary tract cancer and use thereof

The present invention relates to a peptide having an accumulation property specific to biliary tract cancer and use thereof.
This application claims priority based on Japanese Patent Application No. 2015-196227 filed in Japan on October 1, 2015, the contents of which are incorporated herein by reference.

Biliary tract cancer is a cancer composed of bile duct cancer, gallbladder cancer, and duodenal papilla cancer. The number of deaths from bile duct cancer and gallbladder cancer in Japan is about 8,900 men and about 9,300 women in 2013. The number of bile duct cancer and gallbladder cancer cases (national estimates) in 2010 was about 11,300 males and about 11,300 females, accounting for 2% and 3% of the total cancer cases, respectively. Of the patients with bile duct cancer and gallbladder cancer, about 20% of the patients can apply surgery, and the survival rate five years after surgery is about 30%.
Biliary tract cancer has few subjective symptoms at an early stage and may progress to some extent when symptoms such as jaundice, white stool, jaundice urine, abdominal pain appear, and early detection is important in treatment . The biliary tract cancer in the present specification includes bile duct epithelium-derived adenocarcinoma, adenosquamous cell carcinoma, and intrahepatic cholangiocellular carcinoma in terms of histopathology.

Tests and diagnostic methods for biliary tract cancer (particularly intrahepatic or extrahepatic bile duct epithelium-derived cancer) include blood biochemical examination, abdominal ultrasonography, abdominal contrast CT examination, endoscopic retrograde pancreatobiliary imaging (ERCP: Examples include endoscopic retrograde cholangiopancreatography and FDG-PET (Fluorodeoxy glucose-Positron Emission Tomography) inspection.

By the way, in the trend in the medical field utilizing peptides as biomaterials, cell membrane-permeable (cell-absorbing) peptides such as Tat, penetratin, and polyargineine have attracted attention.
However, since these peptides are widely and non-selectively absorbed without distinction between normal cells or normal tissues and tumor cells or tumor tissues, treatment of malignant tumors requiring target-selective drug transport DDS (Drag Delivery) Application to the (System) tool is difficult in that it causes serious side effects. In particular, cell membrane-permeable (cell-absorbing) peptides such as Tat, which are widely used in experimental systems worldwide, are known to cause accumulation in the liver (see, for example, Non-Patent Document 1).
In contrast, cyclic RGD is the only medicinal peptide. Cyclic RGD targets α _v β ₃ integrin, which has been reported to be highly expressed in endothelial cells (and some tumor cells) in new blood vessels or existing blood vessels, and its action point for enhancing vascular permeability. Therefore, it is applied as an imaging agent or a DDS agent in the form of simultaneous use with other drugs, not alone (see, for example, Patent Document 1).

Japanese Patent No. 5721140

In the above-described inspection and diagnosis method for biliary tract cancer (particularly, intrahepatic or extrahepatic bile duct epithelial cancer), the determination criterion of the inspection result is the determination of an abnormal shadow. There is a limit to the accuracy of shadow determination, including the spread of lesions.
In addition, the cyclic RGD described in Patent Document 1 is not a peptide that targets tumor cells and tumor tissue itself, and thus is novel in terms of a peptide having the ability to directly capture cancer. There was still room for improvement.

The present invention has been made in view of the above circumstances, and provides a novel peptide having a specific accumulation property by directly acting on biliary tract cancer cells and tissues.

That is, the present invention includes the following aspects.
[1] The following peptide (a) or (b):
(A) a peptide comprising an amino acid sequence comprising the sequence represented by SEQ ID NO: 1,
(B) a peptide [2] consisting of an amino acid sequence comprising a sequence having an identity of 60% or more with the sequence represented by SEQ ID NO: 1 and having a specific accumulation property for biliary tract cancer [2] a cyclic structure [ 1].
[3] The peptide according to [1] or [2], further comprising cysteine residues at the N-terminus and C-terminus.
[4] The peptide according to any one of [1] to [3], which is a retro-inverso type substituted with a D-form amino acid.
[5] A nucleic acid encoding the peptide according to any one of [1] to [4].
[6] A vector comprising the nucleic acid according to [5].
[7] A carrier comprising the peptide according to any one of [1] to [4].
[8] The carrier according to [7], further comprising a labeling substance or a modifying substance.
[9] The carrier according to [8], wherein the labeling substance is a stable isotope, a radioisotope or a fluorescent substance.
[10] The carrier according to [8] or [9], wherein the modifying substance is a sugar chain or polyethylene glycol.
[11] A pharmaceutical composition comprising the carrier according to any one of [7] to [10] and a physiologically active substance.
[12] The pharmaceutical composition according to [11], which is for treating or diagnosing biliary tract cancer.

According to the present invention, a novel peptide having an accumulation property specific to biliary tract cancer can be provided. In addition, biliary tract cancer can be detected simply, with high sensitivity and selectively.

2 is a fluorescence micrograph of various biliary tract cancer cells to which various peptides in Test Example 1 are added. 4 is a fluorescence micrograph of M213 cells to which Peptide 3 and chlorpromazine in Test Example 2 were added. It is a fluorescence micrograph of M213 cell which added Peptide3 and Dinosaur in Test Example 2. 4 is a fluorescence micrograph of M213 cells to which Peptide 3 and EIPA were added in Test Example 2. It is a fluorescence-microscope photograph of M214 cell which added Peptide3 (L body) in Test Example 3 and was culture | cultivated by different serum concentration. It is a fluorescence-microscope photograph of M214 cell which added Peptide4 (D body) in Experiment 3 and was culture | cultivated by different serum concentration. 4 is a fluorescence micrograph of various biliary tract cancer cells to which Peptide 3 was added in Test Example 4. 6 is a fluorescence micrograph of various liver cancer cells to which Peptide 3 in Test Example 5 was added. 4 is a fluorescence micrograph of various cancer cells to which Peptide 3 in Test Example 6 was added. 7 is a fluorescence micrograph of cells derived from various normal tissues to which Peptide 3 was added in Test Example 7. 6 is a fluorescence micrograph of cholangiocarcinoma cells and normal cells derived from bile ducts co-cultured with Peptide 3 in Test Example 8. It is the fluorescence-microscope photograph of the bright field of the various structures | tissues of the mouse | mouth which transplanted M214 cell in Test Example 9, and administered Peptide3 (L body) intraperitoneally. It is the fluorescence-microscope photograph of the bright field of the various structures | tissue of the mouse | mouth which transplanted the liver which planted the M156 cell in Test Example 10, and administered Peptide3 (L body) intraperitoneally. It is a fluorescence-microscope photograph of the various cells which added Peptide4 (D body) in Experiment 11. It is the fluorescence micrograph of the bright field of the various tissues | tissue of the mouse | mouth which transplanted the TKKK cell in Test Example 12, and administered Peptide3 (L body) or Peptide4 (D body) intraperitoneally. It is a graph which shows the fluorescence intensity in the various structure | tissues of the mouse | mouth which transplanted the TKKK cell in Experiment 12, and administered Peptide3 (L body) intraperitoneally. It is a graph which shows the fluorescence intensity in the various structure | tissues of the mouse | mouth which transplanted the TKKK cell in Experiment 12, and administered Peptide4 (D body) intraperitoneally. It is a graph which shows the fluorescence intensity in the various structures | tissues of the mouse | mouth which transplanted the TKKK cell in Experiment 12, and administered Peptide3 (L body) or Peptide4 (D body) intraperitoneally. It is the fluorescence-microscope photograph of the bright field of the various structures | tissue of the mouse | mouth which transplanted M156 cell in Test Example 13, and administered Peptide4 (D body) intraperitoneally. It is a fluorescence micrograph of a bile duct cancer cell contained in the ascites of a bile duct cancer patient to which Peptide 3 was added in Test Example 14.

[Peptides with specific accumulation properties for biliary tract cancer]
In one embodiment, the present invention provides the following peptide (a) or (b):
(A) A peptide consisting of an amino acid sequence containing the sequence represented by SEQ ID NO: 1.
(B) A peptide comprising an amino acid sequence comprising a sequence having identity of 60% or more with the sequence represented by SEQ ID NO: 1 and having a specific accumulation property for biliary tract cancer.

The peptide of the present embodiment is a novel peptide having an accumulation property specific to biliary tract cancer.

The present inventors have found a novel peptide having an accumulation property specific to biliary tract cancer by an in vitro virus (IVV) method, and have completed the present invention.
In the IVV method, a kind of antibiotic puromycin is bound to the 3 ′ end of mRNA via a PEG (polyethylene glycol) spacer, and cell-free translation reaction is carried out using it as a template, whereby protein and mRNA are purified. A simple mRNA-protein linking molecule IVV covalently linked via is constructed. We constructed an IVV library by creating IVV uniquely. After IVV containing a protein that binds to bait (bait) from this IVV library is picked up in vitro, the mRNA linked to it is reverse-transcribed, amplified by PCR, and the nucleotide sequence is decoded. Thus, interacting proteins can be identified in a very small amount (more than 1000 times the sensitivity of mass spectrometry).

The peptide of this embodiment includes the following peptide (a).
(A) A peptide consisting of an amino acid sequence containing the sequence represented by SEQ ID NO: 1.

The amino acid sequence represented by SEQ ID NO: 1 in the above (a) is a sequence represented by the following amino acid sequence.
LVXGARLVVR (SEQ ID NO: 1)
[In the amino acid sequence represented by SEQ ID NO: 1 above, X is an isoleucine residue (I), an alanine residue (A), an arginine residue (R), a lysine residue (K) or a histidine residue (H ). ]

The peptide (a) has a specific accumulation property for biliary tract cancer. Moreover, even if the peptide of this embodiment is a peptide which consists only of the amino acid sequence represented by sequence number 1, it has accumulation property specific to biliary tract cancer.

In this specification, “biliary tract cancer” means a cancer composed of bile duct cancer, gallbladder cancer, and duodenal papilla cancer bile duct cancer. Bile duct cancer means a malignant tumor that develops from the epithelium of the bile duct. Depending on the site of the bile duct that has developed, hepatic hilar cholangiocarcinoma, distal bile duct cancer, distal bile duct cancer, and liver It can be divided into internal bile duct cancer (bile duct cell carcinoma). Gallbladder cancer means a malignant tumor arising from the gallbladder and gallbladder duct. The duodenal papilla is composed of the papilla bile duct, papilla pancreas, common duct, and large duodenal papilla, and duodenal papilla cancer means cancer that has occurred in the above-mentioned site. In histopathological classification, it refers to adenocarcinoma, adenosquamous carcinoma, and intrahepatic cholangiocellular carcinoma.

As used herein, “accumulation specific to biliary tract cancer” means a property of being highly absorbed and accumulated in biliary tract cancer cells as compared with normal tissues in vivo and tumor cells of other strains. . As shown in Examples described later, it is presumed that the peptide of this embodiment is absorbed into biliary tract cancer cells by clathrin-dependent endocytosis by dynamin.

In the amino acid sequence represented by SEQ ID NO: 1, X is preferably a hydrophilic amino acid residue, and is an arginine residue (R), a lysine residue (K), or a histidine residue (H). Is more preferable, and from the viewpoint of toxicity, an arginine residue (R) is more preferable.

More specifically, among the amino acid sequence represented by SEQ ID NO: 1 in the above (a), the amino acid sequence represented by SEQ ID NO: 2 and the like can be mentioned.
LVRGARLVVR (SEQ ID NO: 2)

The peptide of this embodiment includes the following peptide (b) as a peptide functionally equivalent to the peptide (a).
(B) A peptide comprising an amino acid sequence having an identity of 60% or more with the amino acid sequence represented by SEQ ID NO: 1 and having a specific accumulation property for biliary tract cancer.

In order to be functionally equivalent to the peptide (a) above, it has 60% or more identity. Such identity is preferably 70% or more, more preferably 80% or more, further preferably 85% or more, particularly preferably 90% or more, and most preferably 95% or more.
Furthermore, the peptide of (b) has a specific accumulation property for biliary tract cancer.

Here, the sequence identity of the target amino acid sequence with respect to the reference amino acid sequence can be determined, for example, as follows. First, the reference amino acid sequence and the target amino acid sequence are aligned. Here, a gap may be included in each amino acid sequence so as to maximize the sequence identity. Subsequently, in the reference amino acid sequence and the target amino acid sequence, the number of matched amino acids is calculated, and the sequence identity can be obtained according to the following formula (1).
“Sequence identity (%)” = [number of matched amino acids] / [total number of amino acids in the subject amino acid sequence] × 100 (1)

The peptide (a) or (b) preferably has a cyclic structure. By being a cyclic structure, it is easily absorbed only into biliary tract cancer cells. The peptide of (a) or (b) may be composed of an L-amino acid, a D-amino acid, or a combination thereof, and is preferably a Retro-Inverso type substituted with a D-form amino acid. .
The retro-inverso type makes it difficult to be degraded by intracellular enzymes. The half-life of the peptide (a) or (b) composed only of L-amino acid is 3 to 6 hours, whereas the half-life is increased to 8 to 12 hours due to the Retro-Inverso type. Can do.
L-amino acids are naturally occurring amino acids, and D-amino acids are those in which the chirality of L-amino acid residues is reversed. Moreover, in order to enhance the accumulation property specific to biliary tract cancer, or may be subjected to chemical modification in order to optimize other physical properties.

The peptide (a) or (b) preferably further comprises cysteine residues at the N-terminus and C-terminus. Specific examples include the amino acid sequence represented by SEQ ID NO: 3 below.
CLVRGARLLVRC (SEQ ID NO: 3)
The peptide of this embodiment can take a cyclized form by using disulfide bonds between thiol groups possessed by cysteine residues by providing cysteine residues at the N-terminal and C-terminal.

[Nucleic acid encoding peptide]
In one embodiment, the present invention provides a nucleic acid encoding the peptide described above.

According to the nucleic acid of the present embodiment, a peptide having an accumulation property specific to biliary tract cancer can be obtained.

As a nucleic acid encoding the above peptide, for example, a nucleic acid consisting of the base sequence set forth in SEQ ID NO: 4, or 80% or more, such as 85% or more, such as 90% or more, such as the base sequence set forth in SEQ ID NO: 4, for example Examples include nucleic acids including any combination of base sequences encoding each amino acid that is a component of a peptide having 95% or more identity and specific accumulation properties for biliary tract cancer. The base sequence shown in SEQ ID NO: 4 is a nucleic acid base sequence encoding a peptide consisting of the amino acid sequence of SEQ ID NO: 3 above.

Here, the sequence identity of the control base sequence with respect to the reference base sequence can be determined, for example, as follows. First, the reference base sequence and the target base sequence are aligned. Here, each base sequence may include a gap so as to maximize the sequence identity. Subsequently, in the reference base sequence and the target base sequence, the number of bases of the matched bases is calculated, and the sequence identity can be obtained according to the following formula (2).
“Sequence identity (%)” = [number of matched bases] / [total number of bases of target base sequence] × 100 (2)

[Vector containing nucleic acid encoding peptide]
In one embodiment, the present invention provides a vector comprising the nucleic acid described above.

According to the vector of the present embodiment, a peptide having an accumulation property specific to biliary tract cancer can be obtained.

The vector of this embodiment is preferably an expression vector. The expression vector is not particularly limited. For example, plasmids derived from E. coli such as pBR322, pBR325, pUC12, and pUC13; plasmids derived from Bacillus subtilis such as pUB110, pTP5, and pC194; plasmids derived from yeast such as pSH19 and pSH15; And bacteriophages; viruses such as adenovirus, adeno-associated virus, lentivirus, vaccinia virus, baculovirus, retrovirus, hepatitis virus; and modified vectors thereof.

In the above-described expression vector, the above-mentioned peptide expression promoter is not particularly limited, and examples thereof include animal cells such as EF1α promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, HSV-tk promoter, and the like. Promoters for expression using a host cell such as a promoter for expression, cauliflower mosaic virus (CaMV) 35S promoter, REF (rubber elongation factor) promoter, and insect cells such as polyhedrin promoter and p10 promoter. A promoter for expression used as a host can be used. These promoters can be appropriately selected depending on the host expressing the above-mentioned peptide.

The above-described expression vector may further have a multicloning site, an enhancer, a splicing signal, a poly A addition signal, a selection marker, a replication origin, and the like.

[Career]
In one embodiment, the present invention provides a carrier comprising a peptide as described above.

According to the carrier of the present embodiment, the target substance can be easily and efficiently transported to the biliary tract cancer.

It is preferable that the carrier of this embodiment further includes a labeling substance or a modifying substance. In addition, the carrier of this embodiment may include both a labeling substance and a modifying substance. The labeling substance or modifying substance may be physically or chemically bound to the above-described peptide directly or via a linker. Specifically, the bond may be a coordinate bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any of the known bonds, linkers, and bonding methods can be adopted.

Examples of the labeling substance include stable isotopes, radioisotopes, fluorescent substances, positron emission tomography (PET) nuclide, single photon emission tomography (SPECT) nuclide, nuclide Examples thereof include a magnetic resonance imaging (MRI) contrast agent, a computed tomography (CT) contrast agent, and a magnetic substance. Of these, stable isotopes, radioactive isotopes or fluorescent substances are preferred. By providing the labeling substance, it can be confirmed simply and with high sensitivity whether or not the target substance has been transported to biliary tract cancer.

Examples of stable isotopes include ¹³ C, ¹⁵ N, ² H, ¹⁷ O, and ¹⁸ O. Examples of the radioisotope include ³ H, ¹⁴ C, ¹³ N, ³² P, ³³ P, and ³⁵ S. When the labeling substance is a stable isotope or a radioisotope, the above peptide may be prepared using a stable isotope-labeled amino acid or a radioisotope-labeled amino acid. As amino acids labeled with stable isotopes or radioisotopes, 20 kinds of amino acids (alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine , Valine, tryptophan, cysteine, asparagine, glutamine), and any amino acids included in the above-mentioned peptides are not particularly limited. In addition, the amino acid may be L-form or D-form, and can be appropriately selected as necessary.

The above-mentioned peptide labeled with a stable isotope or a radioisotope is prepared by expressing the above-mentioned vector containing a nucleic acid encoding the above-mentioned peptide in a system in which a stable isotope-labeled amino acid or a radioisotope-labeled amino acid is present. can do. Examples of systems in which stable isotope-labeled amino acids or radioisotope-labeled amino acids are present include cell-free peptide synthesis systems in which stable isotope-labeled amino acids or radioisotope-labeled amino acids are present, live cell peptide synthesis systems, etc. . That is, in a cell-free peptide synthesis system, in addition to a stable isotope-labeled amino acid or a radioisotope-labeled amino acid, a peptide is synthesized using a stable isotope unlabeled amino acid or a radioisotope unlabeled amino acid as a material, or a live cell peptide synthesis system In the above, a cell transformed with the above vector containing a nucleic acid encoding the above peptide is cultured in the presence of a stable isotope labeled amino acid or a radioisotope labeled amino acid to thereby contain the above nucleic acid encoding the above peptide. A stable isotope-labeled or radioisotope-labeled peptide described above can be prepared from a vector.

Expression of the above-mentioned peptide labeled with a stable isotope or radioisotope using a cell-free peptide synthesis system is performed by the above-mentioned vector containing the nucleic acid encoding the above-mentioned peptide, the above-mentioned stable isotope-labeled amino acid or radioisotope In addition to the labeled amino acid, a stable isotope-labeled amino acid or a radioisotope-unlabeled amino acid, a cell extract for cell-free peptide synthesis, which is necessary for the synthesis of the above-mentioned peptide labeled with a stable isotope or a radioisotope, It can be carried out using an energy source (high energy phosphate bond-containing material such as ATP, GTP, creatine phosphate, etc.). Reaction conditions such as temperature and time can be appropriately selected as appropriate. For example, the temperature is 20 to 40 ° C., preferably 23 to 37 ° C., and the reaction time is 1 to 24 hours, preferably 10 to 20 hours.

In the present specification, “cell extract for cell-free peptide synthesis” refers to a translation system involved in protein synthesis such as ribosome, tRNA, or a plant cell, animal cell, which contains components necessary for a transcription system and a translation system, It means an extract from fungal cells and bacterial cells. Specific examples include cell extracts such as Escherichia coli, wheat germ, rabbit reticulocyte, mouse L-cell, Ehrlich ascites tumor cell, HeLa cell, CHO cell, and budding yeast. Preparation of such cell extracts is described, for example, in Pratt, J. et al. M.M. Et al., Transcribation and translation-a practical approach (1984), pp. In accordance with the method described in 179-209, the above cells are disrupted using a French press, glass beads, an ultrasonic disrupter, etc., and a buffer containing several kinds of salts for solubilizing protein components and ribosomes is prepared. In addition, it can be performed by homogenizing and precipitating insoluble components by centrifugation.

The expression of the above-mentioned peptide labeled with a stable isotope or radioisotope using a cell-free peptide synthesis system is, for example, Premium Expression Kit (manufactured by Cell Free Science) with wheat germ extract, Escherichia coli extraction RTS 100, E. You may carry out using commercially available kits, such as E. coli HY Kit (Roche Applied Science company) and a cell-free Quick (manufactured by Taiyo Nippon Sanso Corporation). When the expressed stable isotope-labeled or radioisotope-labeled peptide described above is insoluble, it may be appropriately solubilized using a protein denaturant such as guanidine hydrochloride or urea. The above-mentioned peptides labeled with a stable isotope or a radioisotope are further prepared by fractionation by fractional centrifugation, sucrose density gradient centrifugation, etc., or by purification using affinity column, ion exchange chromatography, etc. You can also

For the expression of the above-mentioned peptide labeled with a stable isotope or radioisotope using a living cell peptide synthesis system, the above-mentioned vector containing a nucleic acid encoding the above-mentioned peptide is introduced into a living cell, and the living cell is nutrientized. In addition to the above-mentioned stable isotope-labeled amino acids or radioisotope-labeled amino acids, stable isotope-labeled peptides or radioisotope-labeled peptides, It can be performed by culturing in a culture solution containing a labeled amino acid or the like. Here, the living cell is not particularly limited as long as it is a living cell capable of expressing the above-described vector containing the nucleic acid encoding the above-mentioned peptide, for example, a mammalian cell line such as a Chinese hamster ovary (CHO) cell, Examples include living cells such as E. coli, yeast cells, insect cells, and plant cells, and E. coli is preferred in view of convenience and cost effectiveness. The expression of the above-mentioned vector containing the nucleic acid encoding the above-mentioned peptide is incorporated into an expression vector designed so that it can be expressed in each living cell by gene recombination technology, and the expression vector is introduced into the living cell. It can be carried out. In addition, introduction of the above-mentioned vector containing a nucleic acid encoding the above-described peptide into a living cell can be performed by a method suitable for the living cell to be used, for example, electroporation method, heat shock method, calcium phosphate method, lipofection Method, DEAE dextran method, microinjection method, particle gun method, method using virus, FuGENE (registered trademark) 6 Transfection Reagent (manufactured by Roche), Lipofectamine 2000 Reagent (manufactured by Invitrogen), Lipofectamine LTX Regent (in vitro) A commercially available transfection reagent such as Lipofectamine 3000 Reagent (manufactured by Invitrogen). Mention may be made of the law and the like.

The above-mentioned peptide labeled with a stable isotope label or radioisotope labeled expressed by a living cell peptide synthesis system crushes or extracts a living cell containing the above-mentioned peptide labeled with a stable isotope or radioisotope. Can be prepared. Examples of the crushing treatment include a physical crushing treatment using a freeze-thaw method, a French press, glass beads, a homogenizer, an ultrasonic crushing device, and the like. Examples of the extraction treatment include extraction treatment using a protein denaturant such as guanidine hydrochloride and urea. The above-mentioned peptide labeled with a stable isotope or a radioisotope is further purified by fractionation using a differential centrifugation method, sucrose density gradient centrifugation, or purification using an affinity column, ion exchange chromatography, etc. It can also be prepared.

Examples of the fluorescent substance include known quantum dots, indocyanine green, 5-aminolevulinic acid (5-ALA; metabolite protoporphyrin IX (PP IX), near-infrared fluorescent dyes (for example, Cy5.5, Cy7, AlexaFluoro, etc.) ) And other known fluorescent dyes (for example, GFP, FITC (Fluorescein), TAMRA, etc.) The above-mentioned peptide labeled with a fluorescent substance is the above-mentioned vector containing a fluorescent substance and a nucleic acid encoding the above-mentioned peptide. May be prepared by the above cell-free peptide synthesis system or living cell peptide synthesis system without using stable isotope-labeled amino acids or radioisotope-labeled amino acids.

Preferably, for example, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁶⁰ Cu, ⁶¹ Cu, ⁶² Cu, ⁶⁷ Cu, ⁶⁴ Cu, ⁴⁸ V as the nuclide for PET and SPECT , Tc-99m, ²⁴¹ Am, ⁵⁵ Co, ⁵⁷ Co, ¹⁵³ Gd, ¹¹¹ In, ¹³³ Ba, ⁸² Rb, ¹³⁹ Ce, Te-123m, ¹³⁷ Cs, ⁸⁶ Y, ⁹⁰ Y, ^185/187 Re, ^186/188 And Re, ¹²⁵ I, or a complex thereof, or a combination thereof. The above-mentioned peptide labeled with the PET nuclide or SPECT nuclide may be prepared by the above-mentioned cell-free peptide synthesis system or the living cell peptide synthesis system containing the above-mentioned vector containing the nucleic acid encoding the above-mentioned peptide.
Examples of the MRI contrast agent, CT contrast agent, and magnetic substance include gadolinium, Gd-DTPA, Gd-DTPA-BMA, Gd-HP-DO3A, iodine, iron, iron oxide, chromium, manganese, or a complex thereof, or a chelate thereof. A complex etc. are mentioned. The above-mentioned peptide labeled with an MRI contrast agent, CT contrast agent or magnetic substance is physically or chemically obtained by directly or via a linker between the MRI contrast agent, CT contrast agent or magnetic substance and the above-mentioned peptide. What is necessary is just to prepare by combining. Specifically, the bond may be a coordinate bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any of the known bonds, linkers, and bonding methods can be adopted.

Examples of the modifying substance include sugar chains and polyethylene glycol (PEG). By providing the modifying substance, the target substance can be easily and efficiently absorbed into the biliary tract cancer cells. The above-mentioned peptide modified with the modifying substance may be prepared by physically or chemically bonding the modifying substance and the above-mentioned peptide directly or via a linker. Specifically, the bond may be a coordinate bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any of the known bonds, linkers, and bonding methods can be adopted.

In the carrier of the present embodiment, the target substance can be appropriately selected depending on the application. For example, when used for imaging biliary tract cancer, the target substance is provided with the above-described labeling substance as described later. In addition, when used in the treatment or diagnosis of biliary tract cancer, as described later, a physiologically active substance can be provided as a target substance. The target substance may be physically or chemically bound to the above peptide directly or via a linker. Specifically, the bond may be a coordinate bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any of the known bonds, linkers, and bonding methods can be adopted.

In the carrier of the present embodiment, when the target substance is a protein, a fusion protein containing the target substance and the above-described peptide can be produced by, for example, the following method. First, a host is transformed with an expression vector containing a nucleic acid encoding a fusion protein. Subsequently, the host is cultured to express the fusion protein. Conditions such as medium composition, culture temperature, time, addition of inducer, etc. can be determined by those skilled in the art according to known methods so that the transformant grows and the fusion protein is efficiently produced. For example, when an antibiotic resistance gene is incorporated into an expression vector as a selection marker, a transformant can be selected by adding an antibiotic to the medium. Subsequently, the fusion protein expressed by the host is purified by an appropriate method to obtain the fusion protein.

The host is not particularly limited as long as it is a living cell capable of expressing an expression vector containing a nucleic acid encoding a fusion protein. For example, a mammalian cell line such as a Chinese hamster ovary (CHO) cell, or a virus (eg, adeno Virus, adeno-associated virus, lentivirus, vaccinia virus, baculovirus, retrovirus, hepatitis virus etc.), microorganisms such as bacteria (eg E. coli etc.), live cells such as yeast cells, insect cells, plant cells Can be mentioned.
Furthermore, in the carrier of this embodiment, an expression vector containing a nucleic acid encoding the above-described fusion protein may be directly introduced into a biliary tract cancer cell or tissue for expression.

[Pharmaceutical composition]
In one embodiment, the present invention provides a pharmaceutical composition comprising the carrier described above and a physiologically active substance.

According to the pharmaceutical composition of this embodiment, biliary tract cancer can be selectively treated.

In the present specification, the “physiologically active substance” is not particularly limited as long as it is effective for treating biliary tract cancer. For example, drugs such as anticancer agents, nucleic acids, antibodies that specifically bind to biliary tract cancer, antibodies Examples thereof include fragments and aptamers.
As the “physiologically active substance”, a molecular target drug having selective cytotoxic activity against biliary tract cancer is preferable. However, since biliary tract cancer is selectively accumulated by the above-mentioned carrier, cytotoxic used as a conventional anticancer agent is used. Medicine may be used.
In addition, the physiologically active substance may be physically or chemically bound to the above carrier directly or via a linker. Specifically, the bond may be a coordinate bond, a covalent bond, a hydrogen bond, a hydrophobic interaction, or a physical adsorption, and any of the known bonds, linkers, and bonding methods can be adopted. Moreover, in the pharmaceutical composition of this embodiment, the above-mentioned carrier may contain the above-mentioned labeling substance or modifying substance.

Examples of the nucleic acid include siRNA, miRNA, antisense, or an artificial nucleic acid that compensates for these functions.

An antibody can be prepared, for example, by immunizing a rodent animal such as a mouse with a peptide derived from biliary tract cancer as an antigen. Further, for example, it can be prepared by screening a phage library. Examples of antibody fragments include Fv, Fab, scFv and the like.

Aptamers are substances that have a specific binding ability to biliary tract cancer. Examples of aptamers include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers having a specific binding ability to biliary tract cancer can be selected by, for example, the systematic evolution of ligand by exponential enrichment (SELEX) method. Peptide aptamers having specific binding ability to biliary tract cancer can be selected by, for example, the two-hybrid method using yeast.

The pharmaceutical composition of the present embodiment can be used for biliary tract cancer diagnosis, biliary tract cancer treatment effect diagnosis, pathological analysis, biliary tract cancer treatment, or diagnosis of disease associated with biliary tract cancer, pathological analysis, treatment, and therapeutic effect diagnosis. . As a diagnostic method, for example, PET, SPECT, CT, MRI, an endoscope, a fluorescence detector, and the like can be used.

<Dose>
The pharmaceutical composition of the present embodiment takes into account the age, sex, weight, symptoms, treatment method, administration method, treatment time, etc. of the test animal (various mammals including humans or non-human animals, preferably humans). Adjust as appropriate.
For example, when the pharmaceutical composition of the present embodiment is injected intravenously (Intravenous: iv) by injection, 5 mg or more per kg body weight in a single administration to a test animal (preferably a human). The amount of peptide is preferably administered, more preferably 5 mg or more and 15 mg or less of peptide, and particularly preferably 5 mg or more and 10 mg or less of peptide.

The number of administration is preferably 1 to several times per week.
Examples of the dosage form include intraarterial injection, intravenous injection, subcutaneous injection, intranasal, intraperitoneal, transbronchial, intramuscular, transdermal, or oral methods known to those skilled in the art. Intravenous injection or intraperitoneal administration is preferred.

<Composition component>
The pharmaceutical composition of this embodiment comprises a therapeutically effective amount of the above-described carrier and bioactive substance, and a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers or diluents include excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners, flavoring agents. Agents, solubilizers, additives and the like. By using one or more of these carriers, pharmaceutical compositions in the form of injections, solutions, capsules, suspensions, emulsions, syrups and the like can be prepared.
A colloidal dispersion system can also be used as the carrier. The colloidal dispersion system is expected to have an effect of enhancing the in vivo stability of the peptide and an effect of enhancing the transferability of the peptide to a specific organ, tissue, or cell. Examples of colloidal dispersion systems include polyethylene glycol, polymer composites, polymer aggregates, nanocapsules, microspheres, beads, oil-in-water emulsifiers, micelles, mixed micelles, and lipids including liposomes. Liposomes and artificial membrane vesicles, which are effective in efficiently transporting peptides to the organs, tissues, or cells, are preferred.

Examples of formulation in the pharmaceutical composition of this embodiment include those used orally as tablets, capsules, elixirs, and microcapsules with sugar coating as necessary.
Alternatively, those which are used parenterally in the form of sterile solutions with water or other pharmaceutically acceptable liquids, or injectable suspensions. Further, a pharmacologically acceptable carrier or diluent, specifically, sterilized water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, Examples thereof include those formulated by mixing with a preservative, a binder and the like, and mixing in a unit dosage form generally required for pharmaceutical practice.

Additives that can be mixed into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic, excipients such as crystalline cellulose, swelling such as corn starch, gelatin, and alginic acid Agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red mono oil or cherry. When the dispensing unit form is a capsule, the above material can further contain a liquid carrier such as fats and oils.

Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride. Suitable solubilizers such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM), HCO-50 may be used in combination.

Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. In addition, a buffering agent (eg, phosphate buffer, sodium acetate buffer, etc.), a soothing agent (eg, procaine hydrochloride, etc.), a stabilizer (eg, benzyl alcohol, phenol, etc.), an antioxidant, etc. are blended. May be. The prepared injection solution is usually filled into a suitable ampoule.

In the case of an injection, it can also be prepared as an aqueous or non-aqueous diluent, suspension, or emulsion as described above. Such sterilization of injections can be performed by blending filter sterilization with a filter, bactericides, and the like. Injectables can be manufactured in the form of business preparation. That is, it can be used as a sterile solid composition by lyophilization, etc., and dissolved in distilled water for injection or other solvent before use.

<Treatment method>
One aspect of the present invention provides a pharmaceutical composition comprising the above carrier and a physiologically active substance for the treatment of biliary tract cancer.
One aspect of the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the above-described carrier and bioactive substance, and a pharmaceutically acceptable carrier or diluent.
One aspect of the present invention also provides a therapeutic agent for biliary tract cancer comprising the pharmaceutical composition.
In addition, one aspect of the present invention provides use of the above-described carrier and physiologically active substance for producing a therapeutic agent for biliary tract cancer.
Another aspect of the present invention provides a method for treating biliary tract cancer, comprising administering an effective amount of the above carrier and physiologically active substance to a patient in need of treatment.

[Method for imaging biliary tract cancer]
In one embodiment, the present invention provides a method for imaging biliary tract cancer using the carrier described above.

According to the method of this embodiment, biliary tract cancer can be detected simply, with high sensitivity and selectively.

In the method of the present embodiment, the carrier described above preferably includes a labeling substance. Furthermore, you may provide the modifier. Examples of the labeling substance and the modifying substance include those described above.

For example, when the above-mentioned carrier with a labeling substance is added to biliary tract cancer cells, the addition amount of the above-mentioned carrier with a labeling substance is preferably 1 μM or more and 4 μM or less in the culture solution. In addition, it can be evaluated whether or not it is accumulated in the biliary tract cancer cells after 30 minutes or more and 3 hours or less after the addition.

In addition, for example, when the above-mentioned carrier comprising a fluorescent substance as a labeling substance is injected intravenously (Intravenous: iv) with an injection, 1 kg body weight can be obtained per administration to a test animal (preferably human). It is preferable to administer a peptide amount of 5 mg or more, more preferably 5 mg to 15 mg of peptide, and particularly preferably 5 mg to 10 mg of peptide.
In addition, for example, when the above-mentioned carrier comprising a stable isotope, a PET nuclide or a SPECT nuclide as a labeling substance is injected intravenously (Intravenous: iv) by injection, the stable isotope used, the PET nuclide Or what is necessary is just to determine dosage from the radiation dose according to the kind of nuclide for SPECT.

In the method of the present embodiment, for example, PET, SPECT, CT, MRI, an endoscope, a fluorescence detector, or the like can be used as a method for detecting the carrier including the labeling substance.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.

[Example 1] Peptide synthesis Protein-RNA chimera type random having a 12 amino acid residue peptide as a phenotype and an mRNA coding sequence as a corresponding genotype via a puromycin that is produced independently Using a peptide library (in vitro virus library; IVVL), each peptide (Peptide 1 to 4) shown in Table 1 below was separated and identified according to a known IVV (in vitro virus) method. In addition, each identified peptide derived from IVVL is synthesized with a FITC (Fluoresceinisothiocynate) label and subjected to hydrochloride treatment. R9 (9-residue continuous D-arginine) is a non-selective membrane-permeable peptide that is currently widely used.
All of these were obtained by consignment synthesis to Sigma Aldrich Japan (Genosis Division).

In addition, the cell lines and origins of various cells used in the following Test Examples 1 to 13 are as shown in Table 2 below. These are what the inventor has maintained by subculturing in the laboratory.

For cholangiocarcinoma cells, MMNK-1 cells, hepatoma cells, HeLa cells, U2OS cells, A172 cells, A549 cells, MCF7 cells, GCIY cells, Lovo cells, BxPC3 cells, KPK cells, PC-3 cells, NHDF cells, 10 Culturing was performed using RPMI1640 medium containing% FBS (RPMI1640 medium). Hepatocyte cells were cultured using 5% FBS-containing hepatocyte medium (Hepatocyte medium).
HPNE cells were cultured using a CS-C medium kit containing 5% FBS. NuLi-1 cells were cultured using BEGM medium (Bronchial Epidermal Growth Medium, Serum-free). Kidney cells were cultured using a growth medium for normal human kidney epithelial cells (RenaLife Comp Kit). TIME cells were cultured using EBM-2-MV Bullet kit (Endothelial Cell Basal Medium-2 Bullet kit).

[Test Example 1] Confirmation test of peptide accumulation in biliary tract cancer cells Peptides 1, 2 and 3 prepared in Example 1 were added to M156 cells, M213 cells, M214 cells and KKU-100 cells in a medium at 4 μM, respectively. It added so that it might become. The cells were incubated for 60 minutes at 37 ° C. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope. Remove the culture supernatant to which the peptide was added before microscopic examination, wash 3 times with 1 × PBS (−), trypsinize, peel off the adherent cells, and immediately transfer to a new 96-well plate. After resuspension, microscopic examination was performed. The results are shown in FIG.

From FIG. 1, fluorescence was detected in all cells for Peptides 1, 2 and 3. In particular, strong fluorescence was detected in cells to which Peptide 3 was added. From the above, it was revealed that Peptide3 has high permeability into cholangiocarcinoma cells.

[Test Example 2] Confirmation test of peptide accumulation in biliary tract cancer cells using inhibitor Peptide 3 or r9 prepared in Example 1 was added to M213 cells so as to be 4 μM in the medium, and the culture was performed at 37 ° C. Incubated for 60 minutes. As an inhibitor for examining the inhibition of peptides into cholangiocarcinoma cells, 20 minutes after the start of culture, 0, 5, 20, 50, 100 μM chlorpromazine or 0, 2, 10, 50, 100 μM 5- ( N-ethyl-N-isopropyl) amiloride (EIPA) was added 30 minutes later and 0, 10, 20, 40, and 80 μM dynaso were added. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIGS. 2A to 2C.
Chlorpromazine is an inhibitor that inhibits clathrin-dependent endocytosis. Dinosaur is an inhibitor that inhibits clathrin-dependent endocytosis by dynamin. EIPA is an inhibitor that inhibits macropinocytosis (phagocytosis).

From FIG. 2A to FIG. 2C, the fluorescence in the cells to which r9 was added decreased with an increase in the amount of EIPA added, whereas the fluorescence in the cells to which Peptide 3 was added increased with an increase in the amount of dynaso added. It became clear that it decreased. From this, it was speculated that the uptake of Peptide3 into cholangiocarcinoma cells was due to clathrin-dependent endocytosis by dynamin.

[Test Example 3] Confirmation test for accumulation of peptides in biliary tract cancer cells in media with different serum concentrations Peptide 3 or Peptide 4 prepared in Example 1 was added to M214 cells in media containing 0%, 10%, 50% FBS. To 4 μM and incubated at 37 ° C. for 60 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIGS. 3A and 3B.
Peptide 3 and Peptide 4 are peptides having the same amino acid sequence, Peptide 3 is composed of only L-form amino acids, and Peptide 4 is composed of only D-form amino acids. That is, Peptide 3 and Peptide 4 are in a mirror isomer relationship.

From FIG. 3A and FIG. 3B, it was confirmed that the cells to which Peptide 4 was added showed stronger fluorescence even after 120 minutes than the cells to which Peptide 3 was added. In addition, even in an environment with a high serum concentration containing 50% FBS, the uptake into cholangiocarcinoma cells by albumin contained in the serum is inhibited, but cells added with Peptide 4 are more cholangiocarcinoma cells. It became clear that the accumulation property of was high. A medium containing 50% FBS is close to the concentration of human blood. Therefore, it was speculated that Peptide 3 and Peptide 4 can be accumulated in cholangiocarcinoma even when added to the human body by intravenous injection or the like.

[Test Example 4] Confirmation test for peptide accumulation in various biliary tract cancer cells Peptide 3 (4 μM) prepared in Example 1 and Hoechst (Dojindo Co., Ltd.) prepared in Example 1 were added to the various biliary tract cancer cells shown in Table 1 above. And cultured at 37 ° C. for 120 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG.

4. From FIG. 4, fluorescence was detected in Peptide3 in M156 cells, M213 cells, M214 cells, KKU-100 cells, and TKKK cells.

[Test Example 5] Confirmation test of peptide accumulation in various cholangiocarcinoma cells Peptide 3 (4 μM) prepared in Example 1 and Hoechst (which is a nuclear stain) were added to the various cholangiocarcinoma cells shown in Table 1 above. (Manufactured by Dojindo Co., Ltd.) was added and incubated at 37 ° C for 120 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG.

From FIG. 5, almost no fluorescence was detected in PLC / PRF / 5 cells and HepG2 cells.

[Test Example 6] Confirmation test for accumulation of peptide in cancer cells derived from various other tissues Peptide 3 (4 μM) prepared in Example 1 and cancer cells derived from other various tissues and M156 cells shown in Table 1 above. Hoechst (manufactured by Dojindo Co., Ltd.), a nuclear stain, was added and incubated at 37 ° C. for 120 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG.

From FIG. 6, almost no fluorescence was detected in GCIY cells and PC-3 cells.

[Test Example 7] Confirmation test for accumulation of peptides in normal cells derived from various tissues Peptide 3 (4 μM) and nuclear stain prepared in Example 1 on normal cells derived from various tissues shown in Table 1 above Hoechst (manufactured by Dojindo Co., Ltd.) was added and cultured at 37 ° C. for 120 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG.

From FIG. 7, no fluorescence was detected in any of the cells.

From Test Examples 4 to 7, it was confirmed that the peptide of the present invention has specific accumulation properties in cholangiocarcinoma cells.

[Test Example 8] Confirmation test of accumulation of peptide in co-cultured bile duct cancer cells and normal cells derived from bile ducts Peptide 3 (4 μM) prepared in Example 1 and M156 cells and MMNK-1 cells co-cultured A medium containing Hoechst (manufactured by Dojindo Co., Ltd.), a nuclear stain, was added and cultured at 37 ° C. for 60 minutes. Simultaneously, Peptide 3 (4 μM) prepared in Example 1 and Hoechst (manufactured by Dojindo Co., Ltd.) as a nuclear stain were added to the co-cultured M156 cells and MMNK-1 cells, followed by incubation at 37 ° C. for 30 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG.

FIG. 8 reveals that Peptide3 is not taken up by MMNK-1 cells but is taken up only by M156 cells. From this, it was revealed that the peptide of the present invention has specific accumulation properties only in cholangiocarcinoma cells, unlike Tat, which is a conventional cell-permeable peptide.

[Test Example 9] Evaluation test of peptide accumulation in various tissues in human cholangiocarcinoma cell transplanted mice NOD-SCID mice injected intraperitoneally (ip) with M214 cells (6 weeks purchased from Charles River Japan) Aged female mice) were prepared as a human leukemia-xenograft model. Subsequently, 150 μg of Peptide 3 prepared in Example 1 was injected intravenously (iv) to a mouse body weight of 20 g. At 90 minutes after administration, the abdomen was opened, and the tumor lesion and peptide distribution were observed under a fluorescent stereomicroscope. The results are shown in FIG. In FIG. 9, “gal” means gallbladder, “live” means liver, “st” means stomach, “sp” means spleen, “br” means brain, “hr” means heart, “kd” means kidney, “lu” means lung, and “tumor” means malignant tumor. In FIG. 9, “Bright Field” is an image taken in a bright field, and “FITC” is an image taken in a dark field. The upper image is an image when the mouse is opened, the lower left image is an image of various extracted tissues, and the lower right image is an image of sliced various extracted tissues. .

From FIG. 9, it was confirmed that Peptide 3 was accumulated only in the gallbladder and malignant tumor.

[Test Example 10] Evaluation test of peptide accumulation in various tissues in mice transplanted with livers transplanted with human cholangiocarcinoma cells NOD-SCID mice (6 weeks purchased from Charles River, Japan) with livers planted with M156 cells Aged female mice). Subsequently, 150 μg of Peptide 3 prepared in Example 1 was injected intravenously (iv) to a mouse body weight of 20 g. At 90 minutes after administration, the abdomen was opened, and the tumor lesion and peptide distribution were observed under a fluorescent stereomicroscope. The results are shown in FIG. In FIG. 10, “gal” means gallbladder, “liv” means liver, “st” means stomach, “sp” means spleen, “br” means brain, “hr” means heart, “kd” means kidney, “lu” means lung, and “tumor” means malignant tumor. In FIG. 10, “Bright Field” is an image taken in a bright field, and “FITC” is an image taken in a dark field. The upper image is an image when the mouse is opened, the lower left image is an image of various extracted tissues, and the lower right image is an image of sliced various extracted tissues. .

FIG. 10 confirmed that Peptide 3 was accumulated only in the gallbladder and malignant tumor.

[Test Example 11] Confirmation test of peptide accumulation in various cells Peptide 4 (4 μM) and nuclear stain prepared in Example 1 were applied to the various biliary tract cancer cells, MMNK-1 cells and Hepatocyte cells shown in Table 1 above. Hoechst (manufactured by Dojindo) was added and incubated at 37 ° C. for 120 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG.

From FIG. 11, fluorescence was detected only in cholangiocarcinoma cells. In addition, when compared with bile duct cancer cells to which Peptide 3 was added, stronger fluorescence was detected in the bile duct cancer cells to which Peptide 4 was added. Furthermore, fluorescence was also detected in SSP25 cells, OCUG-1 cells, TFK-1 cells and HuCCT1 cells in which no fluorescence was detected with Peptide3. From the above, it was confirmed that Peptide4 which is D body has higher accumulation property in cholangiocarcinoma cells.

[Test Example 12] Evaluation test of peptide accumulation in various tissues in human cholangiocarcinoma cell transplanted mice NOD-SCID in which TKKK cells were injected intraperitoneally (ip) and transplanted with a liver in which TKKK cells were transplanted Mice (6-week-old female mice purchased from Charles River, Japan) were prepared as a human cholangiocarcinoma cell transplant model (human leukemia-xenograft model). Subsequently, 150 μg of Peptide 3 or Peptide 4 prepared in Example 1 was injected intravenously (iv) to a mouse body weight of 20 g. At 90 minutes after administration, the abdomen was opened, and the tumor lesion and peptide distribution were observed under a fluorescent stereomicroscope. The results are shown in FIG. In FIG. 12, “gal” means gallbladder, “liv” means liver, “st” means stomach, “sp” means spleen, “br” means brain, “hr” means heart, “kd” means kidney, “lu” means lung, and “tumor” means malignant tumor. In FIG. 12, “Bright Field” is an image taken in a bright field, and “FITC” is an image taken in a dark field. Furthermore, the upper image is an image of a tissue extracted from a mouse added with Peptide3, and the lower image is an image of a tissue extracted from a mouse added with Peptide4. The left image is an image of various extracted tissues, and the right image is an image of sliced various tissues.

From FIG. 12, it was confirmed that Peptide 3 and Peptide 4 were accumulated only in malignant tumors. In addition, stronger fluorescence was detected with Peptide 4 than with Peptide 3.

13A and 13B are graphs showing the ratios of the fluorescence intensities detected in various tissues when the fluorescence detected in each tissue is quantified and the fluorescence detected in the pancreas is 1 in Test Example 12. It is. FIG. 14 is a graph showing the ratio of the fluorescence intensity detected in the malignant tumor when the fluorescence detected in each tissue is quantified and the fluorescence detected in each tissue is 1.
From FIG. 13A and FIG. 13B, in the mouse administered with Peptide3, the fluorescence detected in the malignant tumor was about 8.6 times the fluorescence detected in the liver, whereas in the mouse administered with Peptide4, the malignant tumor was detected. The detected fluorescence was about 45.8 times the fluorescence detected in the liver.
In addition, from FIG. 14, when the fluorescence in any tissue was set to 1, it was detected in the malignant tumor in the mouse administered with Peptide 4 rather than the fluorescence detected in the malignant tumor in the mouse administered with Peptide 3. Fluorescence was higher.

From the above, it was confirmed that Peptide 4, which is D body, has higher accumulation in cholangiocarcinoma cells.

[Test Example 13] Evaluation test of peptide accumulation in various tissues in mice transplanted with livers transplanted with human cholangiocarcinoma cells NOD-SCID mice (6 weeks purchased from Charles River, Japan) with livers planted with M156 cells Aged female mice). Subsequently, 150 μg of Peptide 4 prepared in Example 1 was injected intravenously (iv) to a mouse body weight of 20 g. At 90 minutes after administration, the abdomen was opened, and the tumor lesion and peptide distribution were observed under a fluorescent stereomicroscope. The results are shown in FIG. In FIG. 15, “gal” means gallbladder, “live” means liver, and “tumor” means malignant tumor. In FIG. 15, “Bright Field” is an image taken in a bright field, and “FITC” is an image taken in a dark field. Furthermore, the upper image is an image when the mouse is opened, the lower left image is an image of the extracted liver and malignant tumor, and the lower right image is a slice of the extracted liver and malignant tumor. It is an image of things.

FIG. 15 confirmed that Peptide 4 was accumulated only in the gallbladder and malignant tumor.

[Test Example 14] Peptide evaluation test for peptide accumulation in cholangiocarcinoma cells contained in ascites of patients with cholangiocarcinoma Peptide 3 (4 μM) and nuclear stain prepared in Example 1 in ascites of three patients with cholangiocarcinoma Hoechst (manufactured by Dojindo Co., Ltd.) was added and cultured at 37 ° C. for 120 minutes. Subsequently, the uptake of each peptide in living cells was visually evaluated with an inverted fluorescence microscope by the same method as in Test Example 1. The results are shown in FIG. In FIG. 16, “Phase + Hoechst” indicates a bright field including a nuclear stained image.

From FIG. 16, it was confirmed that Peptide 3 has an accumulation property not only in cultured cells but also in cholangiocarcinoma cells derived from cholangiocarcinoma patients.

According to the present invention, a novel peptide having an accumulation property specific to biliary tract cancer can be provided. In addition, biliary tract cancer can be detected simply, with high sensitivity and selectively.

Claims (12)

1. The peptide of the following (a) or (b).
   (A) a peptide comprising an amino acid sequence comprising the sequence represented by SEQ ID NO: 1,
   (B) a peptide consisting of an amino acid sequence comprising a sequence whose identity to the sequence represented by SEQ ID NO: 1 is 60% or more, and having an accumulation property specific to biliary tract cancer
2. The peptide according to claim 1, which has a cyclic structure.
3. The peptide according to claim 1 or 2, further comprising cysteine residues at the N-terminus and C-terminus.
4. The peptide according to any one of claims 1 to 3, which is a retro-inverso type substituted with a D-form amino acid.
5. A nucleic acid encoding the peptide according to any one of claims 1 to 4.
6. A vector comprising the nucleic acid according to claim 5.
7. A carrier comprising the peptide according to any one of claims 1 to 4.
8. The carrier according to claim 7, further comprising a labeling substance or a modifying substance.
9. The carrier according to claim 8, wherein the labeling substance is a stable isotope, a radioactive isotope or a fluorescent substance.
10. The carrier according to claim 8 or 9, wherein the modifying substance is a sugar chain or polyethylene glycol.
11. A pharmaceutical composition comprising the carrier according to any one of claims 7 to 10 and a physiologically active substance.
12. The pharmaceutical composition according to claim 11, which is for biliary tract cancer treatment or diagnosis.

Images